Mass spectrometry involving ionization by matrix-assisted laser desorption (MALDI) has established itself as a standard procedure for the analysis of biosubstances with large molecules. For this purpose, time-of-flight mass spectrometers (TOF-MS) are usually employed, although Fourier transform ion cyclotron resonance spectrometers (FT-ICR) or radio frequency quadrupole ion trap mass spectrometers (in short: ion traps) have also been utilized.
In the following, the molecules of biosubstances to be studied will be referred to simply as “analyte molecules” or “biomolecules”. In all cases, analyte molecules are present either in very diluted form in aqueous solutions, pure or mixed with organic solvents. Sometimes these analytical solutions are very complex and dirty with respect to the requirements of the analytical procedures, e.g., in the case of body fluids.
The biosubstances include all biopolymers and sometimes other substances with large molecules such as corticosteroids. “Biopolymers” comprise oligonucleotides (i.e. fragments of genetic material in various forms such as DNA or RNA), polysaccharides and proteins (the essential building blocks of the living world) as well as their special analogues and conjugates such as glycoproteins or lipoproteins, and peptides arising from the action of digestive enzymes.
The selection of matrix substance for MALDI depends on the type of analyte molecule; more than a hundred different matrix substances are now known. One of the tasks of the matrix substances is to isolate the analyte molecules from each other wherever possible and bind them to the sample support, to transfer the molecules into the vapor phase by forming a vapor cloud during the laser bombardment, and ultimately to ionize the biomolecules by protonation or deprotonation. For this task it has proven useful to incorporate the analyte molecules individually in the crystals of the matrix substances during their crystallization, or at least finely distributed in the boundary areas between the crystals. Here it seems important to separate the analyte molecules from each other, i.e., no clusters of analyte molecules should be allowed in the prepared matrix crystal sample.
A variety of procedures are known for applying analytes and matrices. The simplest of these entails the pipetting of a solution containing analyte and matrix onto a cleaned, metallic sample support. The drop of solution wets a certain area of the metal surface (or its oxide layer) whose size on hydrophilic surfaces is many times larger than that of the diameter of a drop. The size depends on the hydrophilicity and the microstructuring of the metal surface as well as on the properties of the droplet, in particular that of the solvent. After drying of the solution, a sample spot consisting of small matrix crystals forms that is the same size as that of the originally wetted surface area. The matrix crystals are usually not uniformly distributed throughout the formerly wetted area. As a rule, crystals of the matrix start growing at the inner margin of the wetting surface on the metal plate. They then grow towards the interior of the wetting surface. They often form thin needle crystals, as is the case for example for the frequently used matrices 5-dihydroxybenzoic acid (DHB) or 3-hydroxypicolinic acid (HPA), which often stand out from the carrier plate at the interior of the spot. The center of the spot is frequently empty or covered with fine crystals, although often they cannot be used for MALDI ionization because of their high concentration of alkaline salts. The loading of the crystals with biomolecules is also very uneven. This type of loading therefore requires viewing of the sample support surface during MALDI ionization by a video microscope which can be found in any commercially available mass spectrometer used for this type of analysis. Ion yield and mass resolution vary in the sample spot from place to place. It is often an arduous process to find a suitable position on the sample spot with a satisfactory analyte ion yield and mass resolution, and only experience, trial and error allow for improvements.
Although there are control programs for mass spectrometers with algorithms for automatically seeking the best spots for MALDI-ionization, such procedures, involving many attempts and evaluations, are necessarily very slow.
With other loading procedures the matrix substance is already present on the carrier plate before application of the solvent droplets, which now only contain analyte molecules.
If the surface of the sample carrier plate is not hydrophilic, but hydrophobic, smaller crystal conglomerates are formed, but the droplets tend to wander in an uncontrollable manner during drying. Hence the localization of the crystal conglomerates cannot be predicted and must be sought during the MALDI process. Furthermore, there is a considerable risk that droplets will conglomerate and thus render a separate analysis of samples impossible.
Biosample analyses are now performed in their thousands, a situation which demands automatic high throughput procedures. A visual control or search, or even an automated search, would obstruct such a high throughput procedure.
A procedure has now been developed by the applicant which leads to local and size-defined crystallization fields on small hydrophilic anchor regions of 100 to 800 micrometer in diameter within an otherwise hydrophobic surface (DE 197 54 978 C2). The aqueous drops are fixed by the hydrophilic anchors and prevented from wandering even when they initially rest on surrounding lyophobic areas due to their weight. During drying the droplets withdraw onto the anchor, and relatively dense, homogeneously distributed, crystalline conglomerates arise on the exact position of these anchors (sometimes even structured as a single compact crystalline block depending on the type and concentration of matrix substance). It could be shown that the detection limit for analyte molecules improves with reduction of the surface area of the wetting surface. Thus, smaller quantities of analytes and more diluted solutions can be worked with during sample preparation; such an advantage is expressed in better running biochemical preparatory procedures and reductions in chemical material costs. With a suitable preparation the analytical sensitivity over the surface of the sample is highly uniform. Thus the ionization process can be freed from the need to perform visual or automated searches for favorable sites; instead a “blind” bombardment of the crystal conglomerates with desorbing laser light can be used. This preparation method for prelocated spots of equal sensitivity accelerates the analytical process.
The crystal conglomerates forming on the hydrophilic anchor surfaces reveal a microcrystalline structure suitable for the MALDI-process. As the speed of the drying process is increased, the crystalline structure becomes finer.
Here a “hydrophobic” surface is understood as a water repellant surface, i.e. one resistant to wetting by aqueous solutions. Correspondingly, a “hydrophilic” surface is understood as one that can be easily wetted by water. “Oleophobic” and “oleophilic” (also referred to sometimes as “lipophobic” and “lipophilic”) refer to surfaces which repel or which can be wetted by oil, respectively. Organic solvents that are not miscible with water usually have an oily nature in this meaning of wettability, i.e. they can wet oleophilic faces. They are as a rule miscible with oil. Organic solvents that are miscible with water, e.g. methanol, acetone or acetonitrile, can wet both oleophilic and hydrophilic surfaces in a pure state. However, the wettability of oleophilic surfaces reduces as the water content increases.
An opinion persisted for a long time that hydrophobic surfaces are always also oleophilic, and that oleophobic surfaces are always hydrophilic. However, for some years it has been known that surfaces exist which are both hydrophobic and oleophobic; these include smooth surfaces of perfluorinated hydrocarbons such as polytetrafluoroethylene (PTFE). Such surfaces are designated here as “lyophobic”, a term which has been adopted from colloidal science.
Recently, it has also become known that the wetting or liquid repelling character of a surface strongly depends on its microstructure. An example of this is the so called “lotus effect” (named after the lotus-plant).
The hydrophobicity (oleophobicity, lyophobicity) can be measured essentially by measuring the contact angle which the liquid develops under standardized conditions at the edge of the wetting surface with the solid surface. In an absolute sense a surface of a material is referred to as hydrophobic, oleophobic or lyophobic if the contact angle of the respective liquid level in a capillary constructed from this material is more than 90°. Such a definition is hard to apply to the contact angle of a droplet sitting on a flat surface since the droplet size actually plays a bigger role in this case. In the following, the terms hydrophilc and hydrophobic are not used in an absolute, but rather a relative sense: a surface is more hydrophobic towards a liquid than another surface if the contact angle is larger. In general, a surface is already regarded as hydrophobic if the contact angle is smaller than 90°, but a drop does not run on the surface to form a large wetting surface.
A surface is particularly designated as “hydrophobic” when a drop retracts on a surface during drying or aspiration with a pipette, reducing the wetted surface reduces in size and leaving behind a dry surface (so called “dynamic hydrophobia”).
As a rule, biomolecules are best dissolved in water, sometimes with the addition of organic, water-soluble solvents such as alcohols, acetone or acetonitrile. The analytical solutions of biomolecules sometimes also contain other substances such as glycols, glue-like buffering agents, salts, acids or bases depending on their preparation. The MALDI process is disrupted considerably by the presence of these impurities, sometimes through prevention of protonation, and sometimes through the formation of adducts. In particular, alkali ions often form adducts with analyte molecules of varying size and prevent any precise mass determination. The concentration of alkali ions in the sample preparation, as well as the concentration of other impurity substances must be kept extremely low by careful purification procedures.
For purification and simultaneous enrichment of biomolecules one can use so-called affinity adsorption media similar to those used in affinity chromatography. While in affinity chromatography one uses highly bioselective affinity adsorbents, for the purification of initially unknown mixtures of biopolymers without losses of special types of biomolecules one needs non-specific adsorbents that can bind all biomolecular constituents of the mixture to as near a similar degree as possible.
For purification of peptides, proteins or DNA mixtures, sponge-like microspheres of adsorbent material (such as POROS, a registered trademark of Perseptive Biosystems, Inc.), pipette tips filled with sponge-like adsorbent (such as ZIPTIPs, a registered trademark of Millipore Corporation) or C18 coated magnetized spheres (such as GenoPure, a product of Bruker Daltonics, Inc.) have proven particularly useful until now. These materials are all strongly oleophilic and bind peptides or oligonucleotides via hydrophobic bonds. As a rule, biomolecules can be eluted using aqueous methanol or acetonitrile solutions, and elution can often be assisted by altering the pH-value. However, purification with these materials is labor-intensive since it requires additional materials and additional procedural steps.
Affinity capture methods have become known also for biospecific selection of certain biomolecules in connection with mass spectrometric analysis, see e.g., U.S. Pat. No. 6,020,208, U.S. Pat. No. 6,027,942, or U.S. Pat. No. 5,894,063 (T. W. Hutchens and T.-T. Yip). Such biospecific affinity adsorption processes can be likewise used for purification.
As an alternative or additional procedure one can also remove noxious cations by substitution with ion exchangers. A procedure has also been developed by us to accomplish this (DE 199 23 761 C2).